Nucleosidic phosphorylating agent and methods

ABSTRACT

Compounds of the formula ##STR1## where R is a base labile substituted-phenyloxy protecting group, are useful as phosphorylating reagents in the synthesis of oligonucleotides by a phosphotriester method. Such compounds are made by the reaction of β-cyanoethanol and a corresponding R-phosphorodichloridate. A method of phosphorylating involves reacting such compounds, in the presence of base, with a nucleoside or a polynucleotide having a free 3&#39; hydroxyl group, having the 5&#39;--0 position protected by an organic group labile in acidic medium and having any phosphotriester linking groups protected by an organic group labile in strong basic medium.

BACKGROUND OF THE INVENTION

With the coming of age of recombinant DNA technology attention hasincreasingly been focused on the synthesis of oligonucleotides forvarious purposes, e.g., as hybridization probes for use in locatingcomplementary DNA made by reverse transcription from purified messengerRNA, as primers in the controlled conversion of single todouble-stranded DNA, as plasmidic control regions useful in thebacterial expression of useful proteins, as "linkers" for interpolatingheterologous DNA into plasmids, as genes encoding useful proteins thatthemselves may be bacterially expressed, and so on. In each such caseDNA fragments have hitherto been assembled by condensation ofnucleotides or oligonucleotides according to a sequential plan dictatedby the nucleotide sequence of the desired end product. As one example,the known amino acid sequence of the useful compound somatostatin haspermitted design and synthesis of a corresponding gene, which could thenbe inserted in a bacterial plasmid so as to permit bacterial productionof the protein encoded. K. Itakura, et al., Science 198, 1056-1063(1977).

The construction of oligonucleotides entails phosphorylation of anucleosidic moiety to form the corresponding 3'-phosphate, which is thencondensed with a further, suitably protected nucleosidic moiety to yielda di- or polynucleotide in which the original nucleosidic moieties arelinked by a phosphodiester bridge. In the so-called "triester" methodthe third functionality of the phosphate is protected prior to thecondensation reaction to prevent undue side reactions and to neutralizecharge so as to permit silica gel chromatography techniques in productpurification and recovery. See, e.g., K. Itakura et al, Can. J. Chem.51, 3649-3651 (1973) and the somatostatin work previously referred to. Atypical series of steps in oligonucleotide constructions typical of pasttriester practice may be represented as follows, "B" being thecharacteristic base moiety of the nucleoside involved, X a protectinggroup for the 5'--OH, and R, e.g., p-chlorophenoxy: ##STR2## In theforegoing scheme the possibility exists in the first reaction ofbyproduct formation owing to e.g., multiple phosphorylation. More to thepoint, the intermediate product 3 is charged, so that the β-cyanoethanolreactant in the following step must be used in considerable (e.g., 5X)excess if the presence of a polar nucleotidic moiety is to be avoidedwhen product 4 comes to be purified and, of course, workup andpurification of 4 is in any event complicated by the excess remaining.Finally, end product 5 is itself charged, and hence cannot be purifiedin silica gel chromatography, an otherwise highly convenient tool.

A need has accordingly existed for improved means of DNA and otheroligonucleotide synthesis, so as to diminish recovery losses, otherwiseenhance yields, and to permit more rapid synthesis of key materials inhealth-related and other fields.

BRIEF SUMMARY OF THE INVENTION

The present invention provides as a novel phosphorylating compound thematerial ##STR3## where R is a base labile phenyloxy protecting group.As a single step phosphyorylating reagent, the compound permitsphosphorylation and condensation to go forward without workup ofintermediate product or the need for an interposed β-cyanoethylationstep, and yields as end product a neutral oligonucleotide which admitsof facile purification and recovery in high yields. Using the newreagent, it proved possible in just three months to produce 29 differentoligodeoxyribonucleotides to build genes for human insulin, R. Crea etal, Prod. Nat'l Acad. Sci. USA 75, 5765-5769, Dec. 1978, which couldthen occasion bacterial expression of that precious substance, D.Goeddel et al, Proc. Nat'l. Acad. Sci. 76, 106-110 Jan. 1979; andsubsequently to construct 12 further oligonucleotides to supplysynthetic components of a gene used successfully in the bacterialproduction of human growth hormone, D. Goeddel et al, Nature 281,544-548 (Oct. 1979). These and the other publications referred to hereinare incorporated by reference to illustrate, variously, the backgroundand advantages of the invention. The manner in which the foregoing andother objects and advantages of the invention are attained will furtherappear from the detailed description which follows.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the invention may be made by reaction of β-cyanoethanoland R-phosphorodichloridate in ether/triethylamine, i.e., ##STR4## whereR is a base labile phenyloxy protecting group, e.g., p-chlorophenyloxy,o-chlorophenyloxy, 2,4-dichlorophenyloxy, p-nitrophenyloxy,p-methoxyphenyloxy, etc.

Use of the compounds in DNA synthesis may be represented by thefollowing reaction scheme in which the preferred embodiment of theinvention, p-chlorophenyl-2-cyanoethylphosphorochloridate is employed:##STR5## In the foregoing, Q may be a suitably protected 5' OH (i.e., R₁O-- where R₁ is an organic group labile in acid medium) or, where apolynucleotide is to be phosphorylated and chain extended, a moiety ofstructure ##STR6## where n is zero or an integer from 1 to about 18.

In like fashion, the reactant 10 may alternatively be a polynucleotide,e.g., ##STR7## where m is zero or an integer so chosen that the sum of nand m is not greater than about 17. The group B in any case may be thesame or different and is selected from the group consisting of adenyl,thymyl, guanyl and cytosyl. R₂ is a protecting group that is labile inbasic medium.

The phosphorylation reaction (a) is conducted in a suitable solvent,e.g., acetonitrile, in the presence of base to neutralize hydrogenchloride formed as byproduct, thus driving the reaction towardcompletion. A preferred basic medium for this purpose is1-methylimidazole, which also serves to activate thephosphorochloridate. Temperature is non-critical and all reactionsdepicted may be run at, e.g., room temperature, although thephosphorylation reaction is preferably run at somewhat reducedtemperature, e.g., at or about 0° centigrade. Reaction (b) removes thecyanoethyl moiety by base-catalyzed β-elimination preferably in apyridine/triethylamine/water system (3:1:1 vol/vol). Condensationreaction (c) employs a coupling agent in excess (e.g., 3-4 equivalents),preferably 2,4,6-triisopropylbenzenesulfonyl tetrazolide and isperformed in pyridine, preferably with an excess (e.g., 1.5 equivalentsor more) of the charged reactant 9. Because the intended product 11 isrelatively neutral the excess of the charged reactant 9 may be removedby silica gel chromatography. Thus, as an example, the final reactionmixture is passed through a silica gel column washed first with CHCl₃ toelute side products and coupling agent, then with CHCl₃ /MeOH (95:5vol/vol) to elute the fully protected oligomer, leaving the chargedreactant behind in the column.

Among the many acid labile protecting groups useful in such condensationreactions may be mentioned, e.g., tetrahydropyrenyl,1-methoxycyclohexyl, 4-monomethoxytrityl and, most preferably,4,4'-dimethoxytrityl, which latter may be removed in mild acid medium,e.g., 2% benzenesulfonic acid.

The group R₂ is removable in strong basic medium, e.g., concentratedammonia or NaOH, and may be, e.g., orthochlorophenyl, 2,4-dichlorophenyl, parachlorophenyl, paranitrophenyl, etc. It isaccordingly unaffected by the β-elimination reaction which removes thecyanoethyl moiety, the latter being carried out in weak basic medium,i.e., from near-neutrality to about pH 9.

In the example that follows, the method of forming the most preferredembodiment of the invention is illustrated in greater detail.

EXAMPLE Synthesis of p-chlorophenyl-2-cyanoethylphosphorochloridate.

A solution of freshly distilled triethylamine (14 ml, 0.1 mole) in ether(100 ml) was added, dropwise and under magnetical stirring, to a chilledsolution (ice water bath) of p-chlorophenyl phosphorodichloridate (24.5g. 0.1 mole) and 2-cyanoethanol (7 ml, 0.1 mole) in ether (300 ml).After complete addition of the base, the solution was stirred for onehour at room temperature and then quickly filtrated. The ether wasevaporated off and the product recovered as an oil. Appropriate choiceof the starting phosphorodichloridate yields the other fully protectedphosphorylating agents of the invention, by a like procedure.

I claim:
 1. A method of phosphorylating a moiety of structure ##STR8##wherein B is adenyl, thymyl, guanyl or cytosyl and Q is selected fromthe group consisting of R₁ O-, and ##STR9## wherein n is zero or aninteger from 1 to about 18, R₁ is an organic group labile in acidicmedium and R₂ is an organic group labile in basic medium which comprisesreacting said moiety with a phosphorylating reagent of structure##STR10## wherein R is a base labile phenyloxy protecting group, in thepresence of a weak base to form the product ##STR11##
 2. A methodaccording to claim 1, which further comprises reacting said product inthe presence of base to remove the cyanothyl group and to form thecompound ##STR12##
 3. A method according to claim 2, wherein R₁ is anorganic protecting group labile in acidic medium and R₂ is an organicprotecting group labile in basic medium.
 4. A method according to claim2, wherein R is selected from the group consisting of p-chlorophenyloxy,o-chlorophenyloxy, 2,4-dichlorophenyloxy, p-nitrophenyloxy andp-methoxyphenyloxy groups.
 5. A method according to claim 2, wherein Ris p-chlorophenyloxy.
 6. A method according to claim 2, wherein R₂ is aphenyl group labile in basic medium.
 7. A method according in claim 2,wherein R₂ is selected from the group consisting of p-chlorophenyl,o-chlorophenyl, 2,4-dichlorophenyl, p-nitrophenyl, and p-thiophenylgroups.
 8. A method according to claim 2, wherein R₂ is p-chlorophenyl.9. A method according to claim 2, wherein R₁ is a trityl group labile inacidic medium.
 10. A method according to claim 2, wherein R₁ is4,4'-dimethoxytrityl.
 11. A method according to claim 2, wherein R₁ isselected from the group consisting of 4-monomethoxytrityl and4,4'-dimethoxytrityl groups.